The effect of Cu layer oxidation upon contact with liquid media was investigated by X-ray photoelectron spectroscopy revealing that, after two hours, 55% of Cu atoms are in form of CuO or Cu(OH)2. The Cu-coated nanofibers will be great candidates for wound dressings thanks to an interesting synergistic effect: on the one hand, the rapid release of copper ions kills bacteria, while on the other hand, it stimulates the regeneration with the activation of immune cells. Indeed, copper ions are necessary for the bacteriostatic action of cells of the immune system. The reactive CO2/C2H4 plasma polymers deposited onto PCL-Cu nanofibers can be applied to grafting of viable proteins, peptides, or drugs, and it further explores the versatility of developed nanofibers for biomedical applications use. Copper-coated nanofibrous materials are desirable for catalysis, electrochemistry, sensing, and biomedical use. The preparation of copper or copper-coated nanofibers can be pretty challenging, requiring many chemical steps that we eliminated in our robust approach, where for the first time, Cu was deposited by magnetron sputtering onto temperature-sensitive polymer nanofibers. For the first time, the large-scale modeling of PCL films irradiation by molecular dynamics simulation was performed and allowed to predict the ions penetration depth and tune the deposition conditions. The Cu-coated polycaprolactone (PCL) nanofibers were thoroughly characterized and tested as antibacterial agents for various Gram-positive and Gram-negative bacteria. Fast release of Cu Mainly, the biomedical use of nanofibers attracts the attention of many researchers because they efficiently accelerate wound healing ,12, may olutions , graftinolutions , or by aolutions . In geneEscherichia coli and Gram-positive Staphylococcus aureus bacteria [E. coli and S. aureus bacterial strains [2+ ion release. Phan and co-authors made a similar conclusion for E. coli and B. subtilis [2 embedded into polyacrylonitrile nanofibers would be less effective than CuSO4 but more efficient than metallic Cu thanks to the faster release of Cu2+ ions from oxidized surfaces. However, the authors did not reveal the cytotoxicity of such structures.Polyacrylonitrile (PAN) nanofibers with embedded CuO were tested for antimicrobial breathe masks with high antibacterial effects . PVA nanbacteria . Haider  strains . The mecsubtilis . They alThis work presents a facile, robust, and scalable method for preparing Cu-coated nanofibers based on magnetron sputtering of copper onto the FDA-approved biodegradable polycaprolactone (PCL) nanofibers. The temperature-sensitive polymer nanofibers have never been tested as a substrate for the Cu deposition by magnetron sputtering. The main challenge is the deposition of a well-adhered metallic coating with high Cu content PCL membrane without degradation of the nanofibrous structure. The antibacterial properties against Gram-negative and Gram-positive bacteria and cell viability of mesenchymal stromal cells were studied, and the mechanism of Cu-coated PCL nanofibers onto different strains was discussed.The electrospun nanofibers were prepared by the electrospinning of a 9 wt% solution of polycaprolactone PCL . The processing of the sample can be found elsewhere . Briefly−8 mbar. A 30 sccm flow of a high purity Ar gas (99.99%) was introduced into the deposition chamber, setting the operation pressure to 1.5·10−3 mbar. During the film deposition, the distance between the targets and the substrate was kept at 30 mm and the substrate holder was rotated at 10 rpm to obtain a homogenous film thickness. The deposition time was adjusted to deposit a 50 nm thick film (controlled by deposition onto Si wafer). The Cu-coated nanofibers are referred as PCL-Cu throughout the text.The Cu coatings were deposited by magnetron sputtering of a copper target in an ultra-high-vacuum deposition chamber . The input power to the magnetron was set to 37 W. Before the deposition, the chamber was evacuated down to 6.2·10−3 Pa. The plasma was ignited using radio frequency (RF) power supply Cito 1310-ACNA-N37A-FF  connected to the RFPG-128 disk generator (Beams & Plasmas) installed in the vacuum chamber. The duty cycle and the RF power were set to 5% and 500 W, respectively.The COOH plasma polymer layers were deposited using the vacuum system UVN-2M equipped with the rotary and oil diffusion pumps. The residual pressure of the reactor was below 102 (99.995%), Ar (99.998%), and C2H4 (99.95%) were fed into the vacuum chamber. The flows of the gases were controlled using a Multi-Gas Controller 647C . The flow rates of Ar, CO2, and C2H4 were set to 50, 16.2, and 6.2 sccm, respectively. The pressure in the chamber was measured by a VMB-14 unit  and D395-90-000 BOC Edwards controllers. The distance between RF-electrode and the substrate was set to 8 cm. The deposition time was 15 min and it led to the growth of ~100 nm thick plasma coatings. The plasma coated PCL-Cu nanofibers are referred to as PCL-Cu-COOH throughout the text.COThe microstructure of the nanofibers and the deposited plasma polymers was studied by scanning electron microscopy (SEM) using a Tescan Mira  device. The SEM micrographs were obtained in secondary emission mode with the accelerating voltage of 10 kV and working distance of 9 mm. Micrographs of 1024 × 1024 pixel were acquired. The elemental mappings were obtained using energy dispersive X-ray (EDX) detector .x component at 285 eV.The chemical composition of the sample surfaces was determined by the X-ray photoelectron spectroscopy (XPS) using an Axis Supra spectrometer  equipped with the monochromatic Al Kα X-ray source. The maximum lateral resolution of the analyzed area was 0.7 mm. The spectra were fitted using the CasaXPS software after subtracting the Shirley-type background. The binding energies (BE) for all carbon and oxygen environments were taken from the literature . The BE 2O, respectively, and incubated at 37 °C. After 1, 2, 4, and 24 h, an aliquot of 1 of 500 μL was taken. The sample solution was injected into the plasma through a nebulizer of SeaSpray type using a peristaltic pump with a rate of 0.7 mL/min. Analysis conditions: cooling argon flow—12 L/min, secondary—0.5 L/min; registration time on the first slit—15 s; on the second slit—5 s. The power supplied to the ICP inductor was 1150 W (recommended by the manufacturer of the spectrometer). The registration of emission spectra was carried out at the axial observation of plasma. In the process of sample preparation, the following reagents were used: concentrated nitric acid extra pure, 69.0–71.0% , deionized water purified with the Direct-Q3 system (Millipore) >18 MΩ/cm; high purity argon; single component standard solution—copper (Cu) (Merck). Samples dissolution was performed using concentrated nitric acid with heating ~ 100–150 °C. Sample preparation was performed using disposable plastic tubes with a volume of 5–15 mL, polypropylene container with a volume of 10 mL, and automatic pipette with variable volume . To determine analytes of Cu, the most intense spectral lines were used —328.068, 338.289; 324.754, 327.396 nm, respectively. The validation of the technique by spike experiment was provided.The High-Resolution Spectrometer iCAP 6500  was used. Samples size of 1 × 1 cm were placed in 5 mL of PBS and HThe classical molecular dynamics method in the LAMMPS  software3 cells/well. Additionally, cells were cultivated in 96-well plates in a medium in which the PCL-Cu and PCL-Cu-COOH were soaked (round samples of diameter 0.5 cm in 200 µL) for 1 h and then incubated for 72 h under 5% CO2 atmosphere. A fresh culture medium was added to control cells. After that 5 µL of the MTT solution with the concentration of 5 mg/mL was added to each well, and the plates were incubated for 4 h and then solubilized with a dimethyl sulfoxide solution, as indicated in the manufacturer’s instructions. The optical density was measured with a plate reader Multiskan FC  at the wavelength of 570 nm. The experiment was repeated three times on separate days. For fluorescent microscopic analysis, cells were incubated at 37 °C in the dark with stain solution  for 30 min. Live cells are determined distinguished by the presence of ubiquitous intracellular intense uniform green fluorescence.Cell viability was assayed by the MTT method and fluorescent microscopy. Human mesenchymal stromal cells were extracted from bone marrow using standard methods (the Ethics Committee approved the study of the RICEL-branch of ICG SB RAS (No 115 from 24.12.2015) and cultured in Dulbecco’s modified Eagle’s Medium  that was supplemented with 10% fetal bovine serum . Cells were seeded in 96-well plates on scaffolds (round samples of diameter 0.5 cm) in concentration 7 × 10E. coli ATCC25922, S. aureus ATCC25923, S. Typhimurium ATCC14028, P. aeruginosa ATCC27853 strains were obtained from Remel™, Thermo Fisher Scientific, USA, and Becton Dickenson, France. The bacterial strains were grown in liquid Lysogeny broth (LB) medium at 37 °C for 24 h and then were diluted in saline to give concentrations of 0.675–2.5 × 105 colony-forming units (CFU) mL−1. Antibacterial activity against each strain was determined by the emersion of nanofibers (round samples in diameter 0.5 cm) in a medium volume of 300 µL with bacteria for 24 h. The number of viable microorganisms was estimated via counting of CFU after 24 h of cultivation. All experiments were performed in triplicate.In order to correctly select the conditions where no destruction of such sensitive material as PCL occurs, the computational simulation was performed before the experiments of Cu deposition. The PCL unit cell  was takeTo simulate the irradiation of a film of finite thickness, a PCL slab with a size of 22.7 × 3.94 × 5.31 nm, consisting of 64,800 atoms, was constructed. Periodic boundary conditions were applied in the direction of the y and z axes, and the irradiation was carried out along the x-axis. Before irradiation the slab was relaxed for 100 ps at constant pressure (NPT thermostat), then at a constant temperature (NVT thermostat) equal to T = 300 K.The metal atom was placed randomly at a distance of 1.8 nm from the PCL slab surface, mainly in the center of the YZ plane of the supercell . The atoTo simulate the dissipation of energy into an infinite volume of material, a 0.5 nm thick region was isolated from the side of the PCL slab to the atoms of which temperature control was applied . An NVE thermostat was applied to the remaining atoms of the system, including the metal atom. The simulation continued until the energy of the metal atom exceeded the average thermal energy of the PCL slab.The energies of a swooped Cu atom in the range from 500 eV to 2400 eV were considered. For each selected energy, a series of computer experiments were made, consisting of five simulations. The average values are shown in −2·E0.7739. It is worth noting that in the process of penetration into the PCL, the copper atom has the ability to change the movement direction due to collisions with polymer atoms. Herewith, the penetration angle of the copper atom is random, and the average deviation angle for all simulations is 5.96 degrees.In the energy range under consideration, a power-law dependence of the penetration depth on the atom’s energy is obtained, described by the equation L=4.07·10The SEM micrograph of Cu-coated PCL nanofibrous mat is in The Cu layer covering PCL nanofibers is relatively thick, as sputtering by Ar cluster gun revealed. As shown in 0/Cu+ , copper oxide (II) CuO , copper hydroxide Cu(OH)2  and three Cu(II) satellites centered at 940 ± 0.2 eV, 942.2 ± 0.2 eV and 944.3 ± 0.2 eV. The BE values for all Cu2p and O1s peaks were employed from the literature: Cu0/Cu+ from [2 from [2. Hence, the XPS O1s spectrum of PCL-Cu is completely different as compared to the spectrum of PCL-ref that was fitted by a sum of two peaks: C-O (BE = 533.4 eV) and C = O (BE = 532.2 eV), as shown in To investigate the chemical bonds of copper and other elements presented in the samples, the fitting of high-resolution XPS Cu2p 3/2, C1s, and O1s signals was performed . We analCu+ from ,31, Cu(O [2 from . The curx (BE = 285 eV), carbon neighbored to ester group C-C(O)O (BE = 285.5 eV), ether group C-O (BE = 286.4 eV) and ester group C(O)O (BE = 289.0 eV). The FWHM for all peaks was 1.0 ± 0.1 eV. A sum of only three components fitted the XPS C1s spectrum of PCL-Cu: CHx, C-O and C(O)O O b, where The deposition of the COOH layer of PCL-Cu allows improving the wettability of PCL-Cu nanofibers and the COOH groups play a role of active sites for grafting other compounds. The successful coating was evidenced by significantly different surface composition of COOH-plasma-coated samples (PCL-Cu-COOH) compared to PCL-Cu. As shown in 2+ ions. The stability of Cu layers was inspected visually and by XPS analysis of samples after soaking in PBS and by ICP-OES analyses of PBS and water after PCL-Cu and PCL-Cu-COOH soaking.The antibacterial properties of PCL-Cu and PCL-Cu-COOH samples are expected to be induced by the leaching of CuVery rapid dissolution of Cu layer was evident even by visual inspection as shown in Nevertheless, although no coating was visible for samples after soaking in PBS, XPS analyses revealed that even after 24 h soaking, some amount of Cu still remained at the PCL-Cu-PBS24h sample b,c. Inde2+ ions released in water or PBS at 37 °C is very high and recalculated concentration that is expected to be in the culture media while PCL-Cu cell tests (left Y-axis) exceeds 4.2 μg/mL.As shown in E. coli, S. Typhimurium, P. aeruginosa, and Gram-positive S. aureus. The PCL-ref was used as a negative control and did not have any antibacterial effect. The results are presented in E. coli and S. aureus. In turn against S. Typhimurium and P. aeruginosa scaffolds had no significant antibacterial effect.The antimicrobial activity of nanofibers was studied on the most common pathogenic microorganisms, namely Gram-negative Cells seeded on PCL-Cu and PCL-Cu-COOH did not survive (data not shown). 2+ salts, copper peroxide CuO2, or copper hydroxide Cu(OH)2. The antibacterial properties of the designed materials described in the literature differ significantly. Thus, when comparing the antibacterial action of CuO2 and Cu(OH)2, it was shown that Cu(OH)2 has a significantly lower antibacterial activity compared to CuO2 [E.coli [The antibacterial properties of copper have been known since the ancient period. Now, in the era of antibiotic resistance, researchers again attract much attention, including the development of Cu-based antibacterial materials. Various forms of copper are used: nanoparticles , Cu cont to CuO2 . It is i to CuO2 . In cont [E.coli .E. coli and Gram-positive S. aureus. However, we did not find significant activity against the S. typhimurium and Ps. aeruginosa strains. This phenomenon is most probably related to different sensitivity towards copper for different strains. Bacteria have a number of defense mechanisms against the toxic effects of copper ions: the relative impermeability of the outer and inner membranes of the cell, which leads to a restriction of the intake (sequestration) of copper inside and the inner bacterial membranes to copper ions (intracellular sequestration), metallothionein-like proteins that absorb copper, in the cytoplasm and periplasm and energy-dependent efflux, precipitation as CuS, and extracellular complexation. Also in the cytoplasm and periplasm, some proteins actively remove copper from cells. For S.typhimurium and Ps. aeruginosa the following are known as the next copper-resistant proteins: copper detection proteins , copper efflux proteins  and copper sequestration proteins (CueP for S.typhimurium) [S.typhimurium are 8-16 times higher than for E.coli [Our work demonstrated the very rapid dissolution of the Cu layer from Cu-coated PCL nanofibers and its antibacterial activity against Gram-negative imurium) ,40. The imurium) ,42. It wPs. aeruginosa to copper ions is reported at 44.8 μg/mL, significantly higher than the concentration released from nanomaterials (<10 μg/mL). Hence, possibly, the Cu2+ concentration leached from our PCL-Cu layers is insignificant in inducing the antibacterial effects against S.typhimurium and Ps. aeruginosa.As a result, the MIC of Our work demonstrated that largely different amounts of copper are dissolved by distilled water, phosphate buffer, and culture media. We showed that the highest dissolution rate was visually observed for PCL-Cu and PCL-COOH samples soaked in culture media. The increased rate of copper release in the nutrient medium is most likely associated with the presence of chelating compounds in it, including low-density lipoproteins, proteins , which is the carrier of copper in the body. Using various media, it is possible to control the rate of release of copper ions and, accordingly, the antibacterial effect.2+ release in PBS and water was only 3.5 and 3.6 μg/mL, respectively. Hashmi et al. achieved Cu2+ concentrations up to 33.98 ppm (33.98 μg/mL) for PAN nanofibers with CuO soaked in deionized water during 72 h [2+ antibacterial analysis plays a significant role and affects the results.Molteni et al. compared the release of copper ions in various media . The auting 72 h . Hence, It is worth noting that the dynamics of copper precipitation from the surface also differ. In some cases, there is a prolonged exposure for several days. In our work, similarly as in a majority of other reports , the rapSince the materials developed in our work have a great potential for biomedical use, including regenerative medicine, they must exhibit biocompatibility and activate the regenerative activity of cells. The PCL-Cu and PCL-Cu-COOH were tested for their biocompatibility towards the mesenchymal stromal cells. As mentioned earlier, the rate of copper release from the surface differs significantly depending on the medium. For MSCs cultivation we use standard culture media rich in ions, protein factors, and amino acids, which contribute to the copper extraction and binding. As a result, we have shown that seeded MSCs on PCL-Cu and PCL-Cu-COOH had a high death rate due to the high concentration of copper ions. Since the release of copper ions is fast enough, its concentration is high and cells seeded on fresh PCL-Cu/PCL-CU-COOH nanofibers have a high percentage of death. So in the reference , the con2+ concentration will decrease to the optimal level for cell regeneration. It is known that copper ions are required for collagen synthesis, stimulate VEGF production, and enhance angiogenesis [The influence of copper on tissue regeneration is enormous, thus the nanofibers developed in this work have excellent prospects for future research. Therefore, it will be necessary to check the effect of low pH environment, purulent wounds, with a high hydrogen peroxide content, which may increase the antibacterial activity. In this case, the high rate of copper release plays a positive role, since after cleaning the wound, Cuogenesis . Moreoveogenesis . Since iogenesis ,46. In tE. coli and S. aureus due to a fast release of Cu2+ ions (concentration up to 3.4 µg/mL), and sufficient biocompatibility. Thus, they may demonstrate an interesting synergistic effect when applied for wound healing. On the one hand, the rapid release of copper ions kills bacteria, while on the other hand, it stimulates the regeneration with the activation of immune cells, because copper ions are necessary for the bacteriostatic action of cells of the immune system [2. The reactive COOH groups available at the surface of PCL-Cu coated with carboxyl plasma polymers may be used for grafting viable proteins, peptides, or drugs. It further explores the versatility of developed nanofibers for biomedical applications use.The Cu-coated PCL nanofibrous mats were successfully prepared by using a robust and scalable approach based on Cu magnetron sputter-deposition onto electrospun polymer nanofibers. For the first time, the large-scale modeling of PCL films irradiation by molecular dynamics simulation was performed and allowed to predict the ions penetration depth and tune the deposition conditions. The copper-coated PCL nanofibers exhibited the antibacterial effect against e system . The eff


There has been no assessment of the greenness of the described analytical techniques for the simultaneous determination (SMD) of caffeine and paracetamol. As a result, in comparison to the greener normal-phase high-performance thin-layer chromatography (HPTLC) technique, this research was conducted to develop a rapid, sensitive, and greener reversed-phase HPTLC approach for the SMD of caffeine and paracetamol in commercial formulations. The greenness of both techniques was calculated using the AGREE method. For the SMD of caffeine and paracetamol, the greener normal-phase and reversed-phase HPTLC methods were linear in the 50–500 ng/band and 25–800 ng/band ranges, respectively. For the SMD of caffeine and paracetamol, the greener reversed-phase HPTLC approach was more sensitive, accurate, precise, and robust than the greener normal-phase HPTLC technique. For the SMD of caffeine paracetamol in commercial PANEXT and SAFEXT tablets, the greener reversed-phase HPTLC technique was superior to the greener normal-phase HPTLC approach. The AGREE scores for the greener normal-phase and reversed-phase HPTLC approaches were estimated as 0.81 and 0.83, respectively, indicated excellent greenness profiles for both analytical approaches. The greener reversed-phase HPTLC approach is judged superior to the greener normal-phase HPTLC approach based on numerous validation parameters and pharmaceutical assays. Paracetamol A is the An extensive literature search revealed various analytical approaches for the simultaneous determination (SMD) of caffeine and paracetamol in commercial formulations and biological fluids. For the SMD of caffeine and paracetamol in commercial formulations, different spectrometry techniques involving various chemical procedures, such as derivatization have been used ,7,8,9,10In comparison to the greener normal-phase HPTLC approach, the current study intends to establish and validate a rapid, sensitive, and greener reversed-phase HPTLC approach for the SMD of caffeine and paracetamol in commercial formulations. Following “The International Council for Harmonization (ICH)” Q2-R1 recommendations, the greener normal-phase and reversed-phase HPTLC methods for the SMD of caffeine and paracetamol were validated .v/v), EA/E , EA/E , EA/E , EA/E , and EA/E  were studied as the greener mobile phases. For the development of a suitable band for the SMD of caffeine and paracetamol using the greener normal-phase HPTLC technique, various amounts of ethyl acetate (EA) and ethanol (E), including EA/E , EA/E , EA/E , EA/E , and EA/A  revealed poor chromatographic peaks of caffeine and paracetamol with high asymmetry factor (As) for caffeine (As > 1.25) and paracetamol (As > 1.30). When the greener mobile phase EA/E  was evaluated, it was discovered that this greener mobile phase provided well-resolved and intact chromatographic peaks for caffeine at a retardation factor of (Rf) = 0.40 ± 0.01 and for paracetamol of Rf = 0.59 ± 0.02 (v/v) was chosen as the final mobile phase for the SMD of caffeine and paracetamol in commercial tablets utilizing the greener normal-phase HPTLC method.The greener mobile phases, such as EA/E , E/W , E/W , E/W , and E/W  were studied as the greener mobile phases. All of the green mobile phases investigated were created under chamber saturation conditions , including E/W , E/W , E/W , and E/W  revealed poor chromatographic peaks of caffeine and paracetamol with poor As for caffeine (As > 1.30) and paracetamol (As > 1.35). When the greener mobile phase E/W  was evaluated, it was discovered that this greener mobile phase provided well-resolved and intact chromatographic peaks of caffeine at Rf = 0.43 ± 0.01 and of paracetamol at Rf = 0.57 ± 0.02 (v/v) was chosen as the final mobile phase for the SMD of caffeine and paracetamol in commercial tablets utilizing the greener reversed-phase HPTLC method. The maximum response was obtained at a wavelength of 260 nm for caffeine and paracetamol when the spectral bands for caffeine and paracetamol were recorded using densitometry mode. As a result, the whole SMD of caffeine and paracetamol took place at 260 nm.The greener mobile phases, such as E/W  were found to be 0.9928 and 0.9970, respectively. Caffeine and paracetamol’s regression coefficients (R) were found to be 0.9963 and 0.9984, respectively. The values of R2 and R were highly significant for both the compounds (p < 0.05). These findings suggested a strong link between the concentration and measured response of caffeine and paracetamol. All these findings indicated the reliability of the greener normal-phase HPTLC approach for the SMD of caffeine and paracetamol. The ICH-Q2-R1 guidelines were used to estimate various parameters for the SMD of caffeine and paracetamol . The res2 were found to be 0.9976 and 0.9966, respectively. Caffeine and paracetamol’s R were found to be 0.9987 and 0.9982, respectively. The values of R2 and R were highly significant for both the compounds (p < 0.05). These findings again suggested a strong link between the concentration and measured response of caffeine and paracetamol. All these findings indicated the reliability of the greener reversed-phase HPTLC technique for the SMD of caffeine and paracetamol. However, the greener reversed-phase HPTLC technique was more linear than the greener normal-phase HPTLC technique. The resulting data for the linear regression analysis of caffeine and paracetamol calibration curves utilizing the greener reversed-phase HPTLC technique are summarized in f, As, and number of theoretical plates per meter (N/m) for the greener normal-phase HPTLC technique were determined to be satisfactory. The parameters of the system appropriateness for the greener reversed-phase HPTLC methodology are summarized in f, As, and N/m for the greener reversed-phase HPTLC technique were also determined to be satisfactory.The parameters of the system appropriateness for the greener normal-phase HPTLC methodology are summarized in For assessing caffeine and paracetamol, the percent of recovery was utilized to estimate the accuracy of the greener normal-phase and reversed-phase HPTLC techniques. The accuracy evaluation results for the greener normal-phase HPTLC technique are summarized in The precision of the greener normal-phase and reversed-phase HPTLC techniques was investigated as intra/inter-assay precision and given as a percent of the coefficient of variation (CV) for the SMD of caffeine and paracetamol. f values were also estimated to be 0.39–0.41 and 0.58–0.60, respectively.By introducing slight deliberate modifications in the greener mobile phase components, the durability of the greener normal-phase and reversed-phase HPTLC techniques for the SMD of caffeine and paracetamol was examined. f values were also estimated to be 0.42–0.44 and 0.56–0.58, respectively. These results showed that both analytical techniques were reliable for the SMD of caffeine and paracetamol. For the SMD of caffeine and paracetamol, however, the greener reversed-phase HPTLC approach was more robust than the greener normal-phase HPTLC approach. ± 0.27 and 50.52 ± 0.81 ng/band, respectively. Using the greener normal-phase HPTLC technique, the “LOD and LOQ” for paracetamol were estimated to be 17.05 ± 0.31 and 51.15 ± 0.93 ng/band, respectively. The predicted values of “LOD and LOQ” for caffeine and paracetamol utilizing the greener reversed-phase HPTLC technique are summarized in ± 0.12 and 25.56 ± 0.36 ng/band, respectively. Using the greener reversed-phase HPTLC technique, the “LOD and LOQ” for paracetamol were estimated to be 8.71 ± 0.13 and 26.13 ± 0.39 ng/band, respectively. These data suggested that both analytical techniques were sensitive enough for the SMD of caffeine and paracetamol. For the SMD of caffeine and paracetamol, however, the reversed-phase HPTLC methodology was more sensitive than the normal-phase HPTLC methodology.The “limit of detection (LOD) and limit of quantification (LOQ)” were used to evaluate the sensitivity of the greener normal-phase and reversed-phase HPTLC methods for the SMD of caffeine and paracetamol. The predicted values of “LOD and LOQ” for caffeine and paracetamol utilizing the greener normal-phase HPTLC technique are summarized in f values and superimposed ultra-violet (UV)-absorption spectra of caffeine and paracetamol in the commercial tablets PANEXT and SAFEXT with that of standards caffeine and paracetamol, the specificity of the greener HPTLC approach for the SMD of caffeine and paracetamol was assessed. The overlaid UV spectra of standards caffeine and paracetamol, as well as caffeine and paracetamol in the commercial tablets PANEXT and SAFEXT, are shown in By comparing the Rf data, and wavelengths of caffeine and paracetamol in standards and the commercial tablets PANEXT and SAFEXT.At a wavelength of 260 nm, the maximum densitometric responses of caffeine and paracetamol in standards and the commercial tablets PANEXT and SAFEXT were recorded. The specificity of the greener HPTLC technique for the SMD of caffeine and paracetamol was demonstrated by the identical UV spectra, Rf = 0.40 ± 0.01 for caffeine and Rf = 0.59 ± 0.02 for paracetamol in comparison with those of standards for caffeine and paracetamol using the greener normal-phase HPTLC approach. For the SMD of caffeine and paracetamol in their commercial formulation, the greener normal-phase and reversed-phase HPTLC techniques were used as an alternative to regular analytical approaches. The chromatograms of caffeine and paracetamol from commercial tablets were identified by comparing the TLC spots at Rf = 0.43 ± 0.01 for caffeine and Rf = 0.57 ± 0.02 for paracetamol with those of standards caffeine and paracetamol using the greener reversed-phase HPTLC approach. The chromatograms of caffeine and paracetamol from commercial tablets were identified by comparing their TLC spots at RUsing the greener normal-phase HPTLC technique, the percent assays of caffeine in the commercial PANEXT and SAFEXT tablets were estimated to be 91.23 ± 1.14 and 92.45 ± 1.22 percent, respectively. Using the greener normal-phase HPTLC technique, the percent assays of paracetamol in commercial PANEXT and SAFEXT tablets were estimated to be 89.41 ± 1.04 and 91.13 ± 1.06 percent, respectively. Using the greener reversed-phase HPTLC technique, the percent assays of caffeine in commercial PANEXT and SAFEXT tablets were estimated to be 98.51 ± 1.42 and 101.12 ± 1.53 percent, respectively. Using the greener reversed-phase HPTLC technique, the percent assays of paracetamol in commercial PANEXT and SAFEXT tablets were estimated to be 99.42 ± 1.45 and 100.64 ± 1.49 percent, respectively. The greener normal-phase and reversed-phase HPTLC methods were shown to be suitable for the SMD of caffeine and paracetamol in commercial formulations. However, for the SMD of caffeine and paracetamol in commercial formulations, the reversed-phase HPTLC methodology was more reliable than the normal-phase HPTLC methodology.Various quantitative approaches are available for the greenness estimation of analytical approaches ,38,39,40The standards of caffeine and paracetamol were provided by “Sigma Aldrich ”. HPLC-grades E and EA were provided by “E-Merck ”. The W was obtained from the Milli-Q unit in the laboratory. The commercial tablets PANEXT and SAFEXT were obtained from the pharmacy shop in “Al-Kharj, Saudi Arabia”. All other solvents utilized were of analytical grades.The “HPTLC CAMAG TLC system ” was used for the SMD of caffeine and paracetamol in their standards and commercial tablets. The sample solutions were spotted as 6-mm bands utilizing a “CAMAG Automatic TLC Sampler 4 (ATS4) Sample Applicator ”. The “CAMAG microliter Syringe ” was linked with sample applicator. The application rate for the SMD of caffeine and paracetamol was fixed at 150 nL/s. Under linear ascending mode, the TLC plates were developed in a “CAMAG automated developing chamber 2 (ADC2) ” at a distance of 80 mm. For 30 min at 22 °C, the development chamber was saturated with vapors of greener mobile phases. Caffeine and paracetamol were detected using a wavelength of 260 nm. The slit size (band length × width) and scanning rate were both set at 4 mm × 0.45 mm and 20 mm/s, respectively. Three or six replicates were used for each estimation. The software used was “WinCAT’s ”. v/v); however, in the greener reversed-phase HPTLC approach, the greener mobile phase was E/W .The greener normal-phase and reversed-phase HPTLC methodologies used the same instrumentation and analytical procedures as the normal-phase and reversed-phase HPTLC approaches. The TLC plates and the greener mobile phase components were found to be the most significant differences between the two procedures. In the greener normal-phase HPTLC technique, the TLC plates were glass plates (plate size: 10 cm × 20 cm) pre-coated with normal-phase silica gel (particle size: 5 µm) 60F254S plates, but in the greener reversed-phase HPTLC approach, the TLC plates were glass plates (plate size: 10 cm × 20 cm) pre-coated with reversed-phase silica gel (particle size: 5 µm) 60F254S plates. In both cases, the polymer-binder plate was not used. In the greener normal-phase HPTLC approach, the greener mobile phase was EA/E . For the determination of various validation parameters, three distinct QC samples were prepared freshly.Caffeine and paracetamol stock solutions were made individually by dispensing the requisite amounts of both molecules in the specified amount of respective mobile phase, resulting in a final stock solution of 100 µg/mL for both compounds. The concentrations in the 50–500 ng/band range for caffeine and paracetamol were generated using the greener normal-phase HPTLC methodology and the 25–800 ng/band range for caffeine and paracetamol using the greener reversed-phase HPTLC methodology by diluting variable volumes of caffeine or paracetamol stock solution with the respective mobile phase. For the normal-phase HPTLC methodology, 200 µL of each concentration of caffeine and paracetamol were put to normal-phase TLC plates and reversed-phase TLC plates for the reversed-phase HPTLC methodology. Using both analytical techniques, the spot area of each concentration of caffeine and paracetamol was measured. Caffeine and paracetamol calibration curves were created by graphing the concentrations of both drugs against the observed spot area in six repeats  were weighed and the average weights were computed for the SMD of caffeine and paracetamol in PANEXT and SAFEXT. Each brand’s tablets were coarsely crushed and powdered. A portion of each brand’s powder was dissolved in 100 mL of the relevant mobile phase. For the greener normal-phase and reversed-phase HPTLC methods, 1 mL of this solution of each brand of tablet was diluted again using 10 mL of the corresponding mobile phase. The prepared solutions of PANEXT and SAFEXT commercial tablets were filtered and sonicated for around ten minutes to remove any undissolved excipients. Using the greener normal-phase and reversed-phase HPTLC methods, the generated solutions were used to determine caffeine and paracetamol in commercial tablets PANEXT and SAFEXT.n = 6). For the reversed-phase HPTLC method, the linearity for caffeine and paracetamol was evaluated in the 25–800 ng/band range (n = 6). Utilizing the ICH-Q2-R1 recommendations, the normal-phase and reversed-phase HPTLC techniques for the SMD of caffeine and paracetamol were validated for various parameters . By grapf, As, and N/m was used to evaluate the parameters for the system acceptability for the greener normal-phase and reversed-phase HPTLC techniques for the SMD of caffeine and paracetamol. For both analytical approaches, the Rf, As, and N/m data were computed utilizing their reported equations [The calculation of Rquations .n = 6) was assessed at each QC level. The percent recovery was utilized to examine the accuracy of the normal-phase and reversed-phase HPTLC methods for the SMD of caffeine and paracetamol. For caffeine and paracetamol, the accuracy of the greener normal-phase HPTLC technique was tested at three QC levels: lower QC , middle QC , and high QC . For caffeine and paracetamol, the accuracy of the greener reversed-phase HPTLC technique was tested at three QC levels: LQC (50 ng/band), MQC (300 ng/band), and HQC (800 ng/band). Using both analytical techniques, the percent of recovery for caffeine and paracetamol (n = 6), was used to examine intra-assay variation for caffeine and paracetamol. Quantitation of freshly prepared solutions at LQC, MQC, and HQC on three consecutive days for both analytical techniques (n = 6) was used to investigate inter-assay variation for caffeine and paracetamol.Intra/inter-assay precision was measured for the greener normal-phase and reversed-phase HPTLC methods for caffeine and paracetamol. Quantitation of newly prepared caffeine and paracetamol solutions at LQC, MQC, and HQC on the same day for both analytical techniques (v/v) for caffeine and paracetamol was altered to EA/E  and EA/E  for the greener normal-phase HPTLC technique, and the variations in chromatographic response and Rf values were recorded (n = 6). The greener mobile phase E/W  for caffeine and paracetamol was altered to E/W  and E/W  for the greener reversed-phase HPTLC technique, and the variations in chromatographic response and Rf values were recorded (n = 6).For both analytical techniques, the robustness for caffeine and paracetamol was evaluated by making some slight purposeful modification in the mobile phase composition. The greener mobile phase EA/E  [By using a “standard deviation” technique, the sensitivity of the greener normal-phase and reversed-phase HPTLC approaches for caffeine and paracetamol was examined as “LOD and LOQ”. Caffeine and paracetamol “LOD and LOQ” were computed using their published equations for both analytical procedures (n = 6) .f values and UV spectra of caffeine and paracetamol in commercial tablets PANEXT and SAFEXT were compared with those of standards caffeine and paracetamol to determine the specificity of the greener normal-phase and reversed-phase HPTLC methods for caffeine and paracetamol.The Rn = 3). For both analytical procedures, the quantities of caffeine and paracetamol in commercial tablets were approximated using the calibration curves for caffeine and paracetamol.For the normal-phase HPTLC technique, the obtained solutions of the commercial tablets PANEXT and SAFEXT were put on normal-phase TLC plates and on reversed-phase TLC plates for the reversed-phase HPTLC technique. For all analytical techniques, the chromatographic responses were documented using the identical experimental circumstances employed for the SMD of standards caffeine and paracetamol (The AGREE technique  was utilThe literature lacks greener analytical techniques for the SMD of caffeine and paracetamol. As a result, compared to the greener normal-phase HPTLC approach, this research was carried out to develop and validate the rapid, sensitive, and greener reversed-phase HPTLC approach for the SMD of caffeine and paracetamol in their commercial tablets. For the SMD of caffeine and paracetamol, the greener reversed-phase HPTLC approach is more linear, accurate, precise, robust, and sensitive than the greener normal-phase HPTLC approach. The quantities of caffeine and paracetamol in commercial tablets PANEXT and SAFEXT were found to be significantly higher using the reversed-phase HPTLC methodology compared with the normal-phase HPTLC methodology. The AGREE estimation showed the excellent green properties of both the analytical approaches. For the SMD of caffeine and paracetamol in commercial formulations, the greener reversed-phase HPTLC approach has been presented superior to the greener normal-phase HPTLC approach based on different validation criteria and pharmaceutical assays.


Individuals exposed to chronic adverse experiences in childhood and adolescence are at increased risk of developing neuropsychiatric illnesses such as mood and anxiety disorders. Symptoms of anxiety disorders can often be reduced through exposure therapy, which is based on the process of extinction. Although chronic stress in adolescence is known to exacerbate the impaired extinction of learned fear during this period of development, it remains unclear whether exposure to stressors in adolescence qualitatively affects the mechanisms underlying fear extinction. Brain-derived neurotrophic factor (BDNF) and its principle receptor, tropomyosin receptor kinase B (TrkB), are involved in neuroplasticity underlying fear extinction. The small-molecule TrkB agonist 7,8-dihydroxyflavone  improves fear extinction and reduces fear relapse (reinstatement) in adult mice when administered prior to extinction training but its effects in younger ages are unknown. In this study we tested whether 7,8-DHF enhances extinction retention and leads to less renewal in both stressed and non-stressed adolescent rats. Pre-extinction injection of 7,8-DHF led to lower levels of CS-elicited freezing in both the extinction and conditioning contexts in non-stressed adolescent male rats, but not in those given 7 days of corticosterone. These findings indicate that chronic stress interferes with the effectiveness of pharmacological agonism of TrkB in enhancing fear extinction in adolescence. A greater understanding of the mechanisms underlying extinction in adolescence and the effect of chronic corticosterone exposure on those mechanisms may inform a deeper understanding of the etiology and treatment of pediatric stress-related disorders. Adolescence is often termed a period of “storm and stress” . FurtherOne important process of emotion regulation is extinction of learned fear . A partiD-Cycloserine (DCS) improves subsequent extinction retention in adolescent rats when administered immediately following extinction training . All animals were treated in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes . The Animal Care and Ethics Committee at UNSW Sydney approved all procedures.Subjects were 116 experimentally naïve male Sprague-Dawley rats, bred and housed in the School of Psychology at UNSW Sydney. Rats were maintained in a humidity- and temperature-controlled room on a 12-h light/dark cycle (lights on at 0700). Animals were weaned at postnatal day (P)21-P22 and housed with two or three other rats in plastic boxes (60 cm long × 30 cm wide × 12 cm high) with wire tops . A maximum of one animal per litter was allocated into each experimental group. Water and food were available via computer-based recording software (Blue Iris). Each cabinet also contained a ventilation fan that provided a low level of background noise (∼58 dB). CS and US presentations were controlled by Med-PC V software. The chambers were cleaned with tap water after each experimental session.All behavioral procedures occurred in two sets of chambers . One set of chambers was used as Context A and the other as Context B. Each chamber was fitted with a speaker to deliver a white-noise CS. Chambers were enclosed in sound-attenuating cabinets. Each cabinet was fitted with a camera on the rear wall through which behavior was digitally recorded The two identical chambers referred to as Context A were constructed of stainless-steel walls with a Perspex door and ceiling. The floor consisted of stainless-steel rods spaced 16 mm apart. Underneath the rods was a stainless-steel tray containing corncob bedding. A clear Perspex sheet divided the chamber into two triangular spaces and the rat was placed into the side that housed the speaker. The only sources of lighting in Context A were red LEDs on the ceiling of the cabinet.The Context B chambers were constructed of similar materials to Context A but they differed in terms of size, visual features, lighting, and flooring. Specifically, sheets of paper with 2.5 cm vertical black-and-white stripes covered the outside of the Perspex ceiling and door in these chambers. A clear Perspex sheet covered the grid floor and there was no Perspex divider in the Context B chambers. A white light was placed on top of the chambers to provide additional lighting  to the red light.In experiments for Analysis 2, animals in the chronic stress condition were subcutaneously implanted with a 30 mg 7-day release corticosterone pellet  composed of a proprietary matrix of cholesterol, cellulose, lactose, phosphates, and stearates designed to facilitate continuous diffusion of corticosterone over 7 days . The pellet implantation occurred 5 days before the start of the handling procedures  to ensure animals received 7 days of corticosterone exposure before fear conditioning. Animals housed together were implanted with pellets on the same day. Placebo pellets, purchased from the same supplier, were the same size and consisted of the same matrix without the corticosterone. Dose and duration of hormone administration were chosen based on the average daily dose consumed by rats across 7 days of corticosterone administration in drinking water in our previous studies on extinction in stressed adolescent rats i.e., . Before Rats were given an intraperitoneal (i.p.) injection of 5 mg/kg 7,8-DHF  dissolved in 17% dimethyl sulfoxide  and phosphate-buffered saline  and a scrambled foot-shock US . The US was presented in the last second of the CS so that the stimuli co-terminated. The three CS-US pairings were separated by inter-trial intervals (ITIs) of 135 and 85 s (mean ITI was 110 s). These conditioning parameters were based on those used by Extinction training took place in Context B to minimize the possibility that freezing at extinction could be attributed to learned fear of the context, as opposed to fear of the CS. After a 2-min adaptation period, rats received 30 non-reinforced presentations of the white noise CS .via multiple 10 s presentations . Following a 2-min adaptation period, rats received a 2-min CS presentation.r = 0.94–0.96 across the experiments reported here).Freezing was operationalized as the absence of movement other than that necessary for respiration . Rats wen = 25, 13 injected with 7,8-DHF; included in Analysis 2) or placebo pellet  were weighed on the day of extinction training and following the last behavioral test before euthanasia using carbon dioxide. The animals’ abdomens were cut laterally to expose the kidneys and the adrenal glands were excised from above the kidney with visible fat removed. The adrenal glands were weighed as a pair .A subset of animals implanted with a corticosterone , n = 11 injected with vehicle ] included in Analysis 1 were implanted with a placebo pellet at P28 (±1 day), 5 days before the first day of handling. Analysis 2 compared data collected from rats implanted with a corticosterone pellet at P28 (±1 day), which all underwent the same behavioral procedure 5 days later as in Analysis 1. The aim of Analysis 1 was to examine the efficacy of 7,8-DHF on extinction retention in non-stressed adolescent rats, while the aim of Analysis 2 was to examine the efficacy of 7,8-DHF on rats exposed to chronic corticosterone.The aim of this study was to determine the efficacy of 7,8-DHF in ameliorating the extinction retention deficit in non-stressed adolescent rats and, if so, then test the efficacy of this adjunct in chronically stressed adolescent rats. Two overarching analyses were conducted, each involving pooled data from three experiments with vehicle and 7,8-DHF groups  was included as a factor to detect whether any group main effects or interactions varied by the experimental replication. Pre-CS freezing data at each session was analyzed using ANOVA with group  and experiment as between-subjects factors. CS-elicited freezing during conditioning and extinction were analyzed using separate mixed-model ANOVAs with group and experiment as between-subjects factors and trial or block of six CSs as a repeated measure factor for conditioning and extinction analyses, respectively. When the assumption of sphericity was violated for repeated measure ANOVAs, the Greenhouse-Geisser procedure was followed to adjust degrees of freedom and p values. CS-elicited freezing at the extinction retention and renewal tests were analyzed using separate ANOVAs with group and experiment as between-subjects factors. Given that renewal can be viewed as the degree of relapse outside the extinction context, a subsequent mixed-model ANOVA compared freezing across groups across tests, with test as a repeated measures factor. Interactions were explored with simple main effects. Bodyweight and adrenal gland weight as a percentage of bodyweight were analyzed with 2 × 2 ANOVAs with factors of drug  and pellet (placebo or corticosterone). Measures of effect sizes are also given  or had failed to learn during extinction  was excluded from the analysis. This resulted in the exclusion of nine rats from the 7,8-DHF group in Analysis 1, four rats from the 7,8-DHF group in Analysis 2, and one rat from the vehicle group in Analysis 2. In addition, the extinction data of three rats in the 7,8-DHF group and the renewal results of three rats in the control group of Analysis 1 were not included in the analysis due to experimenter error . Furthermore, three of the rats that had their adrenal glands excised did not have their weight recorded at 2 days post-pellet washout due to errors in weight recording.F = 2.38, p = 0.074, ηp2= 0.090, extinction block by pellet interaction; vehicle group: largest F = 2.38, p = 0.13, ηp2= 0.071, pellet effect for conditioning pre-CS]. Therefore, the subsequent analyses disregarded whether animals had pellets or not.We initially compared the behavioral data of those implanted with a placebo pellet to those not implanted with a pellet in rats injected with 7,8-DHF or vehicle. These analyses confirmed that placebo pellet implantation did not affect behavior during any pre-CS period, conditioning, extinction, extinction retention or renewal , extinction training , the extinction retention test , or the renewal test . An effect of experiment or interaction of experiment with group was not detected at conditioning, the extinction retention test, or the renewal test . Pre-CS freezing in the 7,8-DHF and vehicle groups varied at extinction training across experiments  such that the pre-CS freezing was slightly higher in the vehicle controls (M = 10.28) relative to the 7,8-DHF group (M = 1.25) in one out of three experiments . Overall, these results suggest that pre-CS freezing was relatively low across most sessions and was largely unaffected by group.F = 122.70, p < 0.001, ηp2 = 0.687] but no group or experiment effects or interactions .F = 48.34, p < 0.001, ηp2= 0.477], but no main effects of group  or experiment , nor an interaction of block by group . The effects of block and group did not vary by experiment . Whilst a block by group by experiment interaction was detected , follow-up ANOVAs with simple main effects examining group differences across block separately across experiments did not reveal any meaningful differences in the rate of extinction between 7,8-DHF or vehicle groups; the 7,8-DHF group (M = 20.56) had significantly lower freezing than the vehicle group (M = 51.24) only at block 4 in one experiment (p = 0.033). Overall, these results indicate that 7,8-DHF did not affect average levels of CS-freezing or the rate of extinction.A mixed-model ANOVA of the extinction training data detected a block main effect . The group effect was consistent across experiments . This suggests that 7,8-DHF improved extinction retention in non-stressed adolescent rats.F = 7.40, p = 0.009, ηp2= 0.122, see F = 2.42, p = 0.10, ηp2= 0.084; group by experiment interaction: F = 1.42, p = 0.25, ηp2= 0.051].There was a significant difference in level of CS-elicited freezing between groups, with those injected with 7,8-DHF exhibiting lower levels than those injected with vehicle, suggesting less renewal in the 7,8-DHF-treated group , indicative of renewal, and a main effect of drug , but no significant test by drug interaction . These results confirm that, on average, 7,8-DHF reduced post-extinction freezing but suggest that both 7,8-DHF and vehicle groups had a comparable degree of renewal of fear outside of the extinction context.Given that renewal can be quantified as the degree of relapse when performance is tested outside of the extinction training context, a subsequent analysis examined whether each group had significant changes in freezing from the retention test (Context B) to the renewal test (Context A). A mixed-model ANOVA on CS-elicited freezing was conducted with drug  as a between-group factor and test  as a within-subjects factor. This analysis revealed a main effect of test , extinction training , extinction retention test , or renewal . Furthermore, there were no effects of experiment or group by experiment interactions during pre-CS freezing at conditioning, extinction training, extinction retention test, or renewal .As shown in F = 58.16, p < 0.001, ηp2 = 0.624] with no group or experiment effects or interactions being detected .F = 17.29, p < 0.001, ηp2= 0.331] and an experiment effect . However, Tukey’s post hoc tests on the experiment main effect did not detect any significant differences in average freezing across experiments . No effect of group or interactions were detected .A mixed-model ANOVA of the extinction data detected a block main effect . This suggests that 7,8-DHF did not improve extinction retention in chronically stressed adolescent rats. While there was no effect of experiment , there was a significant group by experiment interaction . This interaction was further explored by simple main effects, which found no significant effect of group within each experiment , suggesting that group differences within experiments were not significant.As shown in F = 1.30, p = 0.26, ηp2= 0.036, see F = 1.15, p = 0.33, ηp2= 0.062; group by experiment interaction: F = 0.13, p = 0.72, ηp2= 0.004].The groups did not differ in level of CS-elicited freezing on the renewal test , indicative of renewal, but no main effect of drug  or drug by test interaction . These results suggest that both 7,8-DHF and vehicle groups had a comparable degree of renewal of fear outside of the extinction context.As in Analysis 1, a subsequent mixed-model ANOVA examined whether each group had significant changes in CS-elicited freezing from the retention test (Context B) to the renewal test (Context A). This ANOVA had drug  as a between-group factor and test  as a within-subjects factor. This analysis revealed a main effect of test .Adrenal weights differed between groups, with the animals implanted with corticosterone pellets  1 h before extinction training facilitated extinction retention (as indicated by lower levels of CS-elicited freezing) in both the extinction and the conditioning contexts in non-stressed adolescent rats . However, 7,8-DHF administration had no impact in chronically stressed adolescent rats . These results contrast with those of via slow release implanted pellets). Therefore, it is clear that chronic corticosterone exposure diminishes the effectiveness of at least one behavioral and two pharmacological approaches to enhancing extinction retention in adolescence.Past research has shown that, compared to juvenile and adult animals, non-stressed adolescents have diminished extinction retention . HoweverOne potential explanation for why chronic corticosterone reduces the maintenance of fear extinction in adolescent rats is that such exposure downregulates subunits of the NMDA receptors within critical brain regions that are necessary for extinction consolidation. The activation of NMDA receptors and their downstream signaling cascades  are crucial for the protein synthesis underlying the formation of long-term memories, such as extinction memories, at least in adult animals . NMDA reAs 7,8-DHF did not improve extinction retention in adolescent rats exposed to chronic stress, it is important to consider alternate means by which extinction retention can be improved in this population. In line with this, another area for future research is the examination of the efficacy of 7,8-DHF following extended extinction training in animals exposed to chronic stress. While non-stressed adolescents demonstrate good extinction retention following extended extinction training, those that have been exposed to chronic stress continue to exhibit poor extinction retention even following extended extinction training . This suin vivo, including activation of adenosine receptors  agonist , and theeceptors . In addieceptors  and thateceptors . Furthervia these slow-release pellets had a physiological effect on the adolescents in these experiments.Another limitation of the experiments reported here is that no measures of stress hormone levels in the blood of the adolescent rats were taken in order to confirm that the corticosterone pellet implantation did indeed increase circulating corticosterone levels. However, measures of adrenal glands that were taken 4 days following the cessation of corticosterone exposure show that chronic corticosterone exposure resulted in significantly reduced adrenal weights, replicating past studies with chronic exogenous corticosterone administration in the drinking water of adolescent male rats e.g., . In addivia the implantation of corticosterone pellets). While exposure to chronic elevated levels of corticosterone does indeed lead to behavioral and neural changes that also occur following other chronic stress induction procedures (A third limitation of these studies derives from the way animals were exposed to chronic stress (ocedures , an animocedures . TherefoFuture work may also seek to extend the present work in male adolescent rats by testing whether 7,8-DHF enhances fear extinction consolidation in adolescent females and whether chronic stress interferes with such an effect. Not only are fluctuations in estradiol levels across the rodent estrous cycle associated with varying effectiveness of extinction in adolescent female rats  but 7,8-The experiments described here demonstrate that whilst 7,8-DHF facilitates extinction retention in male non-stressed adolescents it does not facilitate extinction retention in adolescents exposed to chronic stress, at least when the same extinction conditions are used. These results add to the broader literature which has demonstrated that two other approaches that facilitate extinction retention in non-stressed rats, DCS and extended extinction, do not facilitate extinction retention in those exposed to chronic stress. These results provide further insight into the etiology and treatment of pediatric stress-related disorders, and call for further research into the mechanisms underlying the extinction retention deficit in chronically stressed adolescents, and for methods by which this deficit can be ameliorated.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Animal Care and Ethics Committee at UNSW Sydney.AS collected all the data and wrote the first draft of the manuscript. AS and KB did all the statistical analyses. KB and RR edited the manuscript and provided funding for the study. All authors contributed to the conceptualization and design of the study, worked on subsequent drafts of the manuscript, and agreed on the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The addition of nanomaterials, such as graphene and graphene oxide, can improve the mechanical properties of hydroxyapatite (HA) nanocomposites (NCPs). However, both the dispersive state of the starting materials and the sintering process play central roles in improving the mechanical properties of the final HA NCPs. Herein, we studied the mechanical properties of a reduced graphene oxide (r-GO)/HA NCP, for which an ultra-high shear force was used to achieve a nano-sized mixture through the dispersion of r-GO. A low-temperature, short-duration spark plasma sintering (SPS) process was used to realize high-density, non-decomposing r-GO/HA NCPs with an improved fracture toughness of 97.8% via the addition of 0.5 wt.% r-GO. Greater quantities of r-GO improve the hardness and the fracture strength. The improved mechanical properties of r-GO/HA NCPs suggest their future applicability in biomedical engineering, including use as sintered bodies in dentistry, plasma spray-coatings for metal surfaces, and materials for 3D printing in orthopedics. Recently, implant biomaterials have been studied closely for their tissue engineering applications . Due to Hydroxyapatite (HA) is a biomaterial commonly used in surgical operations . Due to The mechanical properties of ceramic materials can be improved by adding a particulate second phase ,21. The 2, may cause HA to decompose during high-temperature manufacturing processes, resulting in a reduction in the biological activity [Although the mechanical properties of HA can be enhanced by the additional second phase, the inherent biological properties of HA and the activity of the adjacent tissue could be damaged by the addition of a second phase. In addition, some reinforcing materials, such as ZrOactivity . Previouactivity ,25,26; hSince the discovery of graphene, its unique material properties in the context of electrical, thermal, optical, and mechanical applications have greatly impacted different research fields, such as electronics and biomedicine ,28,29. SThe r-GO  of 300–800 nm with a horizontal thickness of 0.7–1.2 nm was fabricated via the improved Hummer method. By reducing the graphene oxide using a microwave process, we can remove the functional groups and restore the carbon structure. The HA powders with a particle size of 60 nm were obtained from Yu-Ching Co., Hsinchu, Taiwan. The r-GO/HA mixture was achieved by mixing 3 g r-GO in powder form and 200 g HA in powder form into 100 mL and 500 mL of N-Methylpyrrolidone (NMP) solvent, respectively. The mixture was then treated in an ultra-high-pressure homogenizer for 5 cycles of the dispersion treatment. The ultra-high shear force required for mixing r-GO and HA was generated using the ultra-high-pressure homogenizer (NVL-ED) from Yoshidakikai Co., Nagoya, Japan. Via normal dispersion processing, the performance quality of the dispersed particles may be reduced due to the damaged particle surface. Here, we used wet processing, wherein the slurry was directed to the nozzle through a high-pressure plunger pump. The maximum flow rate was 290 mL/s when the maximum processing pressure was 200 MPa. Homogeneous dispersion can be achieved by controlling the power, which enables very fine distribution. The nozzle used here was a cross (X-type) nozzle. This nozzle design requires large amounts of processing energy and is mainly used for crushing and dispersion processing. The material of the nozzle was single crystalline diamond so as to avoid contamination and severe wear.θ range of 0–90° on a high-resolution X-ray diffractometer. Transmission electron microscopy (TEM) was performed using a JEOL JEM-2100 TEM . Raman measurements were taken using a micro-Raman system  with a helium–neon laser as the excitation source, operating at a 633 nm wavelength and equipped with a 40× objective lens. Scanning electron microscopy (SEM) was conducted using a JEOL JSM-7800 F field-emission SEM . The thickness of the r-GO was measured using a non-contact Fizeau laser interferometer .The X-ray diffraction (XRD) patterns of the NCP were obtained in a 2Spark plasma sintering (SPS)  is a solBending strength analyses were performed with a universal testing machine . The test samples of 10 bars for each NCP were prepared by filling a graphite mold with a diameter of 50.8 mm with r-GO/HA. After a circular cake-shaped sample was formed via SPS, the surface was trimmed with a surface grinder, and the sample was then cut with a very slow cutting machine. The tested samples had a length of 35 mm, a width of 4 mm, and a height of 4 mm. The sintered body was tested with a UTM to measure the compressive and bending strengths using a pressing fixture and a three-point bending fixture, at 1.5 mm/min and with a maximum down force of 300 kg.−1, indicating that the number of graphite layers is less than five [han five . Previouhan five  have proThe r-GO homogenized at an ultra-high pressure was added into the HA solution, which was again mixed via ultra-high-pressure homogenization with various adding ratios of r-GO concentration . Five different concentrations of r-GO solution  were assessed for their effects on mechanical properties. The r-GO solutions, all of which were homogenized at ultra-high pressures, were individually mixed with an HA solution. The newly formed r-GO/HA mixture was then homogenized again at an ultra-high pressure to ensure uniform mixing. After the mixing process, the slurry was vacuum-distilled to remove the solvent, and the dried r-GO/HA NCPs were ground and sieved (screen mesh: 100 mesh). The powder after sieving is shown in The SPS temperature was set at 1000 °C, and the pores were encapsulated via vacuum evacuation (less than 10 Pa). At this point, the upper and lower electrodes applied a pre-pressure of 25 MPa to the sinter in the mold, and then started electric heating. The SPS temperature was increased to 600 °C, then held for 5 min. This was to ensure that any organic impurities from previous processes were completely evaporated, as well as to avoid forming closed pores during the pre-pressing process. After the temperature was held at 600 °C for 5 min, the pressure of the upper and lower electrodes was increased to 50 MPa and the temperature was increased to 1000 °C at a heating rate of 100 °C/min. After holding the temperature at 1000 °C for 10 min, the samples were cooled in a vacuum to below 150 °C. The sinters were then removed for subsequent analysis and mechanical performance testing.The two-inch diameter sinters were further ground to obtain a smooth surface that could facilitate the measurements of true density and porosity as the r-GO is added. The density and porosity of the sintered samples were measured via the buoyancy method following the Archimedean principle, the boiling water method, the wax-sealing method and the vacuum saturation method. The measured results are shown in X-ray diffraction (XRD) analyses were carried out on the sintered r-GO/HA NCPs . The resAfter measuring the densities and porosities of the sinters, they were molded into an elongated shape with the following dimension: 35 mm length, 4 mm width and 4 mm height. The surface of the sinters was then abraded with sandpaper in running water. Lastly, the surface was polished using diamond paste to minimize the possible defects that could significantly alter the measurement data. The three-point flexural failure strengths of the sintered specimens were tested under the conditions of 1.5 mm/min and a maximum down force of 150 kg, using a UTM with a down-compression fixture and a three-point anti-folding fixture. The results are shown in A load test force of 0.5 Newtons was employed to measure the Vickers hardness of the sintered specimens. A previous study showed that the Vickers hardness increases with an increasing load . At low IC, can be calculated using the median crack system [1/2, which is 97.8% higher than that of pure HA. When the amount of r-GO was increased to 5 wt.%, the fracture toughness reached 3.89 MPa m1/2, which is 24.2% higher than that of pure HA. Increasing the amount of r-GO first helps increase the fracture toughness; however, when the addition of r-GO exceeds 0.5 wt.%, a crack appears along the grain boundary and a so-called epitaxial failure forms, and intragranular failures also arise that split the crystal grains. Furthermore, the addition of r-GO graphene may have a bridging effect on the cracks.Through the result of the measurement of Vickers hardness, the fracture toughness, Kk system ,(1)KIC=From the mechanical measurement data, we can infer that the three mechanical properties reach their highest values when the amount of graphene added is 0.5–1 wt.%; however, the values of the mechanical properties begin to deteriorate as the amount of r-GO added exceeds 1 wt.%. When the amount of added r-GO is less than 1wt.%, the r-GO between the HA particles inhibits the growth of HA crystal grains, such that the sintered body exhibits grain refinement and the cracks progress. The process gives rise to twists and turns, as shown in We have successfully fabricated high-density, non-decomposing r-GO/HA NCPs with improved fracture toughness. Ultra-high shear force was used to realize the nano-sized r-GO that was dispersed in the HA matrix. The low-temperature, short-duration spark plasma sintering process was used to achieve r-GO/HA NCPs with fracture toughness of 97.8% via the addition of 0.5 wt.% r-GO. The improved mechanical properties of the r-GO/HA NCPs suggest this process’ applicability in biomedical engineering, such as its use for sintering bodies in dentistry, plasma spray-coating on metal surfaces, and 3D printing in orthopedics.


Duchenne muscular dystrophy (DMD) is the most common and cureless muscle pediatric genetic disease, which is caused by the lack or the drastically reduced expression of dystrophin. Experimental therapeutic approaches for DMD have been mainly focused in recent years on attempts to restore the expression of dystrophin. While significant progress was achieved, the therapeutic benefit of treated patients is still unsatisfactory. Efficiency in gene therapy for DMD is hampered not only by incompletely resolved technical issues, but likely also due to the progressive nature of DMD. It is indeed suspected that some of the secondary pathologies, which are evolving over time in DMD patients, are not fully corrected by the restoration of dystrophin expression. We recently identified perturbations of the mevalonate pathway and of cholesterol metabolism in DMD patients. Taking advantage of the mdx model for DMD, we then demonstrated that some of these perturbations are improved by treatment with the cholesterol-lowering drug, simvastatin. In the present investigation, we tested whether the combination of the restoration of dystrophin expression with simvastatin treatment could have an additive beneficial effect in the mdx model. We confirmed the positive effects of microdystrophin, and of simvastatin, when administrated separately, but detected no additive effect by their combination. Thus, the present study does not support an additive beneficial effect by combining dystrophin restoration with a metabolic normalization by simvastatin. The only routinely used medication for DMD patients is glucosteroid drugs, which can at best only slightly delay the disease progression [Duchenne muscular dystrophy (DMD) is the most common inherited pediatric muscle disorder. It is an X-linked genetic progressive myopathy characterized by muscle wasting and weakness, which leads to loss of motor functions, cardiac and respiratory impairment, and premature death . DMD occgression , however2 + level, perturbed nNOS signaling, TGF beta signaling and fibrosis, energy metabolism dysregulation, lipid accumulation, calcification, necrosis, and inflammation [Such experimental therapeutic approaches, based on gene therapy, cell therapy and drug discovery, are focused on the restoration of dystrophin expression ,5,6,7. Dammation ,8,9. It In 2015, the Froehner’s group demonstrated a substantial improvement of the functional parameters of skeletal muscles in the mdx mouse that was treated by the cholesterol-lowering drug, Simvastatin . This imIn a recent study, we profiled circulating miRNAs in a large cohort of DMD patients. We developed a new approach for the interpretation of miRNA dysregulation, which predicted that the mevalonate pathway and cholesterol metabolism are dysregulated in DMD . Treatinn = 6), as compared to a healthy C57Bl/10 control group. The comparison groups included: (1) control healthy C57Bl/6 (WT), (2) untreated mdx (mdx), (3) mdx treated with Simvastatin only (mdx Simva), (4) mdx treated with AAV-microdystrophin at low titer (mdx µ-dys-low), (5) mdx treated with AAV-microdystrophin at low titer plus simvastatin (mdx µ-dys-low + simva), and (6) mdx treated with AAV-microdystrophin at high titer (mdx µ-dys-high)  mdx were randomly assigned to five treatment groups (ys-high) a.13 viral genome (vg)/kg, which is, in our experience, required to transduce close to 100% of the myofibers in the TA and the GA muscles in the young adult mdx. The low titer that was selected (the 4th group), of 5.0 × 1012 vg/kg, (a 1/6 of the high titer), is the dose that in our knowledge and experience required for the transduction of below 25% of the myofibers of the TA and GA muscles in mdx, which is below the threshold for a measurable muscle function benefit. This experimental design was made in order to test whether the combination of a low dose with a boosting drug (simvastatin here) (in the 5th group) could improve the dystrophic tissue and/or provide functional benefit to the dystrophic muscle to a level, close to the high titer (6th group) treated group. Simvastatin was mixed into a standard rodent diet at a concentration of 80 mg/kg, which is a dose within the normal range prescribed for humans, based on mouse-to-human equivalence calculations, and that demonstrated therapeutic efficacy in the mdx model [The viral titer that was selected for the high titer group (the 6th group) of 3 × 10dx model . Simvastdx model . Muscle Rplp0) in the Tibialis Anterior (TA), muscle presented a proportionally dose-responsive increased expression from AAV-untreated, to the low and high-dose AAV injected animals, (Vector copy-number (VCN) quantification . A small reduction, of microdystrophin mRNA level in the simvastatin treated group  was detected also in the GA muscle (p ≤ 0.072), but not in the diaphragm, where simvastatin treatment did not affect the mRNA level of the µ-dystrophin transgene  was reduced, on the limit of significance, compared to µ-dys-low treatment alone . However, such reduction  was not detected in the GA muscle , of 20 ± 4.19% dystrophin positive fibers was detected in the mdx µ-dys-low + Simva group  muscles detected a dose-dependent increased expression from the low to the high-titer mdx groups, and a reduced microdystrophin protein level in the combined therapy group (mdx µ-dys-low + Simva) compared to µ-dys-low treatment alone  was elevated in untreated mdx compared to the wild-type and presented an expected dose-responsive effect with AAV-microdystrophin treatment: mdx µ-dys-low only partially restored fibrosis staining while mdx µ-dys-high brought it back to the wild-type level a,b. PrevAnalysis of myofiber damage was assessed by IgG staining, which is nonspecifically attached to necrotic myofibers . In agrep = 0.037)  a. Finall= 0.037) b. The tr= 0.037) c, which,= 0.037) d. High tRecently, we found strong evidence for the perturbations of cholesterol metabolism in DMD . In agreFor simplicity, we may interpret the results in the order of (1), the effect of the microdystrophin only, followed by (2), the effect of simvastatin only, and finally (3), the effect of their co-administration. Accordingly, in (1), the mouse groups that were not subjected to simvastatin treatment (microdystrophin-only groups), as expected, VCN b and dysIn (2), the analysis of the simvastatin-only group, we observed a reduced diaphragm fibrosis level a,b and aLastly, it is of particular interest to interpret the effect of the combined therapy, by comparing the groups of low-dose microdystrophin with and without simvastatin (groups 4 and 5). Concerning microdystrophin expression: while no effect of simvastatin was observed on the infectivity (VCN) of the viral vectors, a small reduction in the expression of the microdystrophin was detected, consistently at the mRNA and protein levels, in the simvastatin treated group c–g. ConcThus, when administrated alone, simvastatin treatment improved diaphragm fibrosis, which however was not improved by the co-administration of simvastatin on top of microdystrophin treatment. Similarly, the myofiber damage indicators mCK, Myom−3 and IgG positive fibers, did not improve or even get worst (mCK) by the co-administration of simvastatin on top of microdystrophin treatment.Altogether, the data may suggest a mixed effect of simvastatin on the dystrophic parameters in the mdx model. On the one hand, consistently, simvastatin reduces diaphragm fibrosis. On the other hand, simvastatin might reduce slightly the expression of the dystrophin transgene , and simvastatin alone, or in combination with microdystrophin, seems to increases the proportion of necrotic myofiber. Consequently, the muscle force tests did not indicate functional improvement by the co-administration of simvastatin, on top of the beneficial effect of the microdystrophin alone. Thus, we concluded that the co-administration of simvastatin does not potentiate the benefit of gene therapy in the mdx mouse model for Duchenne muscular dystrophy.Study limitations: The Simvastatin treatment regime of the present study is identical to the 2015 study of the Froehner group , and to Another limitation of the present study is the lack of characterization of the molecular mechanism of the reduced expression of the microdystrophin transgene under the condition of simvastatin treatment. This surprising observation shall be the subject of future investigations.In summary, the present investigation does not support the combination of simvastatin with the gene-therapy restoration of dystrophin. While the results of the present combined approach are disappointing, the rationale for the quest for efficient complementary treatment in DMD, remained solid. An ongoing study in our group is focused on other approaches for metabolic normalization in DMD, to be combined with the restoration of dystrophin expression.C57Bl10 (WT/BL10) and mdx (C57BL/10ScSn-Dmdmdx/J) mice were obtained from Charles River laboratories, Miserey, France. Mice were housed in an SPF barrier facility with a 12-h light/dark cycle, and were provided with food and water ad libitum. In this study, only male mice were used. For the in vivo studies, the operators who performed vector delivery, sample and tissue collection, and functional analyses were blinded to the treatment groups.https://safe-lab.com/safe_en/ accessed on 1 December 2021) at a concentration of 80 mg/kg. Seven week-old mice were treated for seven weeks. Muscle biopsies were dissected, snap-frozen in isopentane in liquid nitrogen, and processed for RNA, protein and histological analyses.Simvastatin was mixed into a standard rodent diet  construct is our optimized version of the microdystrophin that was developed by the laboratories of Jeffrey Chamberlain (H2mDys)  and GeorHEK293-T cells, cultured in suspension, were transfected with the three plasmids coding for the adenovirus helper proteins, the AAV Rep and Cap proteins, and the ITR-flanked transgene expression cassette. Three days after the transfection, cells were harvested, chemically lysed, treated with benzonase , and filtered. Viral capsids were purified by affinity chromatography, formulated in sterile PBS, and the vector stock was stored at −80 °C. Titers of AAV vector stock were determined by using quantitative real-time polymerase chain reaction (qPCR). Viral DNA was extracted using the MagNA Pure 96 DNA and viral NA small volume kit  according to the manufacturer’s instructions. PCR was performed in ABI PRISM 7900 HT Sequence Detector with Absolute ROX mix , using the ITR-specific primers, forward 5′-CTCCATCACTAGGGGTTCCTTG-3′, reverse 5′-GTAGATAAGTAGCATGGC-3′ and the probe 5′-TAGTTAATGATTAACCC-3′.DNA was extracted from transversal sections of the TA muscle, using the NucleoMag Pathogen kit . qPCR for the detection of vector number was performed in duplicates with the LightCycler 480 system , using the SYBER Green technology according to the manufacturer’s instruction, with the forward primer (5′-GATTGAGAAGCTGCTGGACC-3′) and the reverse primer (5′-TCTGGTGGTGTAGCTGGAAG-3′), producing a 191 bp band.Proteins were extracted with RIPA lysis buffer  complemented with Protease Inhibitor Cocktails  and Benzonase . Total protein was quantified with Pierce™ BCA Protein Assay (Thermo Fisher Scientific). Protein extracts were separated on 3–8% Tris-Acetate polyacrylamide gel (Thermo Fisher Scientific) and transferred to a nitrocellulose membrane using the iBlot2 Dry Blotting system (Thermo Fisher Scientific). Dystrophin expression was detected with a mix of Dys-B (Leica: NCL-DYSB) diluted 1:200, and Dys−2 (Leica: NCL-DYS2), diluted 1:50, and normalized to GAPDH intensity, using the Odyssey technology (Thermo Fisher Scientific) scanner and Image Studio Lite 4.0 software .p-values were calculated by a one-way ANOVA.Dissected tissues were frozen in isopentane in liquid nitrogen. Transverse 8µm cryosections of the TA muscle were air-dried and stored at −80 °C. Slices were rehydrated 5 min in PBS, fixed 5 min in 3.7% ice-cold methanol-free PFA, permeabilized in 0.1% Triton-X100 for 5 min, and blocked 30 min in 10% goat serum in PBS (blocking buffer) and additional 30 min in Mouse-on-Mouse IgG 1× Blocking Solution. Sections were incubated with 1:50 primary antibody (Leica: NCL-DYSB) diluted with PBS in 10% blocking buffer overnight at 4 °C. Samples were washed twice in PBS and incubated for 45 min at room temperature (RT) with Alexa-conjugated secondary antibodies , 1:1000 in 10% blocking solution at a dark humid chamber. Samples were washed three times in PBS and mounted in DAPI Fluoromount-G . Images were digitalized using Axioscan Z1 slide scanner  under a Plan-Apochromat 10×/0.45 M27 dry objective  and using a digital CMOS camera ORCA-Flash4.0 . The size of a pixel is 0.65 µm. Tile scan images were reconstructed with ZEN software . Total and positive fibers were manually counted on four random images (900 × 900 pixels) per transversal section, using the ImageJ software. Sirius-red stained transversal sections were scanned with the AxioScan  10 ×. The images were analyzed by morphometry with the HISTOLAB  software.Blood samples were collected by retro-orbital bleeding and quickly centrifuged for 10 min at 8000 rpm. Sera were harvested and further centrifuged to completely remove cell contaminants. The sera were finally stored at −80 °C until measurement. Ten μL of mouse serum was used to colorimetrically measure creatine phosphokinase concentration by FUJI DRI-CHEM nx500 system (DMV Imaging).TM BCA Protein Assay . Protein extracts were separated on 4–12% Bis-Tris polyacrylamide gel (Thermo Fisher Scientific), and transferred to a nitrocellulose membrane using the iBlot2 Dry Blotting system (Thermo Fisher Scientific) for 8 min 30 at 20 V. Total protein on blotted membranes were determined with the Revert 700 Total Protein  staining. Membranes were washed, blocked in Odyssey Blocking Buffer (LI-COR), 1 h, room temperature. Primary antibody  was incubated overnight at 4 °C. After washings in 1× TBST (Tris/HCl (pH 7.5—20 mM)/NaCl (150 mM)/Tween 20 (0.1%)), the diluted secondary antibodies (1/1000 in 50% Odyssey blocking buffer) were incubated 1 h at room temperature. Membrane images were acquired with the Odyssey Infrared Imaging System. Band density was quantified using Image Studio Lite 4.0 software  and normalized to total protein values.Sera protein concentration was measured with Piercehttps://www.bioseblab.com/ accessed on 1 December 2021; France Grip Test 25N). Three independent measurements were performed, and the mean value of weight-normalized grip strength was calculated.The motor capacity of the mice was evaluated using the escape and the 4-limb grip tests. The procedures follow the recommendations of TREAT NMD SOP for the DMD animal models . Brieflyp < 0.05; ** p < 0.01, *** p < 0.005, **** p < 0.001). The Rstudio and GraphPad PRISM 7.01 program  was used for statistics except for the data in


The EQ-5D is a generic measure of health that is widely applied for health economic and non-economic purposes. Population norms can be used to facilitate the interpretation of EQ-5D data. The objective of this study was to develop a set of pooled normative EQ-5D-3L values for the five largest European economies (EUR5).EQ-5D-3L index values based on the time trade-off (TTO) were available for all EUR5 countries : France, Germany, Italy, Spain, and the United Kingdom (UK). Country-specific data sets were aggregated and weighted to facilitate the derivation of norms for gender and age groups. Analyses included equal weighting and weighting by population and economy size. Norms were also calculated using the European visual analog scale-based value set (European VAS), the EQ VAS and separately by dimension.Pooled mean (SD) population weighted TTO values for males/females were 0.967 (0.122)/0.959 (0.118) for ages 18–24; 0.965 (0.096)/0.954 (0.117) for ages 25–34; 0.943 (0.165)/0.936 (0.169) for ages 35–44; 0.934 (0.150)/0.921 (0.157) for ages 45–54; 0.896 (0.188)/0.875 (0.197) for ages 55–64; 0.900 (0.158)/0.839 (0.218) for ages 65–74; and 0.830 (0.234)/0.756 (0.291) for ages 75 and older. Mean values decreased and variance increased with age; females had slightly lower mean values than males across all age bands. The unequal weighting approaches produced similar point estimates with smaller variances. Mean values for the European VAS were slightly lower than those for the TTO-based index.Normative EQ-5D-3L values can be used to benchmark the outcomes of treated patients against the health of the general population. EUR5 norms may be useful in research applications inferring to Europe or the European Union as a whole, particularly when sample size precludes analysis at the country level. The EQ-5D is a generic measure of health that is applied in economic evaluations of health care interventions as well as for non-economic purposes as a health outcome measure. Applicable to a wide range of health conditions, it provides a simple descriptive profile and a visual analogue scale (EQ VAS) that can be used for assessing population health, evaluating health outcomes in clinical trials, and for routine outcomes measurement in health systems. The EQ-5D profile data can be converted into an index value (‘utility’) for health status for use in the economic evaluation of health care. Preference-based generic measures of health such as the EQ-5D and the Health Utilities Index (HUI) are recommended sources of utilities for cost-utility analysis by the Second Panel on Cost-effectiveness in Health and Medicine , but alsDue to its widespread use and evidence supporting its validity for many applications, the 3-level EQ-5D (EQ-5D-3L) is often used as the source of utilities to inform cost-effectiveness models –8. BeyonGeneral population-based survey data for France (N = 2892), Germany (N = 2032), Italy (N = 4709), Spain (N = 5473), and the UK (N = 6319) were identified in which measures completed by respondents included the EQ-5D-3L. Data sources included the ESEMeD  dataset , the Wort und Bild Verlag survey (Germany), and the 2014 Health Survey for England, using the English population as a proxy for the UK –14. For EQ-5D-3L consists of a descriptive health state classifier and a self-rating of “your health today” using the EQ VAS . Each ofNorms were calculated for three types of summary scores. First, for each population dataset, country-specific time trade-off (TTO)-based value sets were applied to calculate index-based utility values  –20. Secow = 0.20) and weighting by population size and economy size using per capita Gross Domestic Product (GDP) [n is the sample size, s2 the variance, c the number of countries.First, norms were calculated by age and gender for each country separately. Subsequently, country-specific data sets were aggregated and weighted per country, to derive the EUR5 norms overall, and for gender and age-based subgroups. By weighting the five country-specific index value set data, both the norm data and the preference structure of the five countries were combined into a pooled EUR5 set of TTO-based index values. We included equal weighting  . Populatct (GDP) –27 , followed by anxiety/depression (13%) Table . An age Normative EQ-5D-3L values can be used to benchmark the outcomes of treated patients against the health of the general population. EUR5 norms may be useful in research applications inferring to Europe or the European Union as a whole, particularly when sample size precludes analysis at the country level. Population norms representing the EUR5 could facilitate a broader approach to identify unmet need based on clinical efficacy outcomes and establish burden of illness in Europe. For these purposes we recommend the population weighted set of norm values  group recently reported normative scores across 14 European countries, Canada and US to facilitate interpretation of scores in cancer , 32. TheThis study has several strengths and limitations. The data used in this study were based on studies conducted at different time periods. Although each population-based study was rigorously designed and collected data elements that enabled the generation of normative data by age and gender, population health in certain countries might have changed over time, as well as preferences that are reflected in the value sets. Several approaches to assigning country-specific weights were used, but the results were similar whether equal weights were applied or not, i.e., based on size of population and economy, which is indicative of a robust pooling approach for our purposes. Note that variance was smaller with unequal weighting approaches, which is due to the fact that we applied weights at the country level . ConsideA complicating issue in estimating normative values for the EUR5 countries is how Brexit will affect the UK and the European Union as economic entities. However, as the results show, trends in normative values are similar across countries and are primarily driven by age and gender. Note that the UK was still part of the European Union at the time this study was conducted and at the time of data collection. It might well be that the EUR5 ‘construct’ will continue to be useful for social, economicor other purposes in the foreseeable future.In summary, normative EQ-5D-3L values were derived that can be used to benchmark the outcomes of patients against the health of the general population. EUR5 norms were generated for utilities based on country-specific TTO value sets, for a European-based value set estimated from VAS values, and for self-reported VAS scores. These normative values may be useful in research applications inferring to Europe or the European Union as a whole, particularly when sample size precludes analysis at the country level.